The primary objective of the proposed research is to provide a comprehensive understanding of the functions of the Saccharomyces cerevisiae Snf1 protein kinase. The two Specific Aims are exploratory in nature and are designed to identify, using proteomic and genomic approaches, proteins that are phosphorylated by and genes that are regulated by Snf1. Snf1 is a critical component of a signal transduction cascade that orchestrates cellular responses to changes in the availability of glucose and other nutrients in yeast. Snf1 is highly related, both structurally and functionally, to the human AMP-activated protein kinase, AMPK. AMPK, as a sensor of the metabolic conditions within cells, has direct links to diabetes, obesity, cancer, and heart disease. Using Snf1 as a model, we seek to identify the full spectrum of proteins and genes regulated by these important metabolic and nutritional sensors. Specific Aim 1 is to identify direct protein substrates of Snf1. The approach is to identify changes in protein phosphorylation, at a genome-wide level, that occur upon rapid inactivation of Snf1. These experiments will employ genetically modified Snf1 derivatives that are specifically susceptible to chemical inhibitors or conditionally destroyed by proteolysis. Two-dimensional difference gel electrophoresis combined with mass spectrometry will be used to identify phosphorylation changes among cellular proteins. A second goal of Aim 1 is to test directly whether the phosphorylation of RNA polymerase II is regulated by Snf1, a prediction based on our genetic observations. Specific Aim 2 is to identify genes whose transcription is directly influenced by Snf1. The approach is to perform DNA microarray studies on yeast strains that are sensitized to Snf1 function. In one case, strains will express Snf1 derivatives that can be rapidly and conditionally inactivated. In a second case, strains will express a gain-of-function mutation in SNF4, a positive regulator of Snf1. A second goal of Aim 2 is to establish an in vitro transcription assay to test whether Snf1 can directly regulate the RNA polymerase II transcription machinery. The proposed studies have direct relevance to the human condition. Remarkably, AMPK has roles in diabetes, obesity, cancer and heart disease, some of the most common and deadly threats to public health. The conservation between Snf1 and AMPK and between their potential targets strongly suggests that the proposed studies will provide new insights into the function of AMPK. [unreadable] [unreadable] [unreadable]